JPH01313502A - Production of powdery sodium hyaluronate - Google Patents
Production of powdery sodium hyaluronateInfo
- Publication number
- JPH01313502A JPH01313502A JP14536888A JP14536888A JPH01313502A JP H01313502 A JPH01313502 A JP H01313502A JP 14536888 A JP14536888 A JP 14536888A JP 14536888 A JP14536888 A JP 14536888A JP H01313502 A JPH01313502 A JP H01313502A
- Authority
- JP
- Japan
- Prior art keywords
- precipitate
- sodium hyaluronate
- water
- organic solvent
- soluble organic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920002385 Sodium hyaluronate Polymers 0.000 title claims abstract description 95
- 229940010747 sodium hyaluronate Drugs 0.000 title claims abstract description 95
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 title claims abstract description 95
- 238000004519 manufacturing process Methods 0.000 title claims description 16
- 239000002244 precipitate Substances 0.000 claims abstract description 74
- 239000003960 organic solvent Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 41
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 18
- 239000012452 mother liquor Substances 0.000 claims abstract description 17
- 238000001035 drying Methods 0.000 claims abstract description 13
- 239000011780 sodium chloride Substances 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 7
- 238000000967 suction filtration Methods 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000011085 pressure filtration Methods 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 29
- 238000005406 washing Methods 0.000 abstract description 9
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 abstract description 2
- 230000001376 precipitating effect Effects 0.000 abstract description 2
- 239000011734 sodium Substances 0.000 abstract description 2
- 229910052708 sodium Inorganic materials 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 17
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 14
- 229920002674 hyaluronan Polymers 0.000 description 14
- 229960003160 hyaluronic acid Drugs 0.000 description 14
- 238000004090 dissolution Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 238000000926 separation method Methods 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 238000000605 extraction Methods 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 159000000000 sodium salts Chemical class 0.000 description 4
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 235000002867 manganese chloride Nutrition 0.000 description 2
- 239000011565 manganese chloride Substances 0.000 description 2
- 229940099607 manganese chloride Drugs 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 235000010265 sodium sulphite Nutrition 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 235000019345 sodium thiosulphate Nutrition 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 210000004127 vitreous body Anatomy 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 208000036487 Arthropathies Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000264435 Streptococcus dysgalactiae subsp. equisimilis Species 0.000 description 1
- 241000194048 Streptococcus equi Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 101100162169 Xenopus laevis adrm1-a gene Proteins 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- 238000007922 dissolution test Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- -1 impropatol Chemical compound 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 235000019691 monocalcium phosphate Nutrition 0.000 description 1
- 229910000150 monocalcium phosphate Inorganic materials 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、水への溶解性の良好なヒアルロン酸ナトリウ
ムの製造法に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing sodium hyaluronate having good solubility in water.
ヒアルロン酸は多糖類の一種であシ、関節、硝子体、調
帯、軟骨、皮膚、鳥類のトサカなどの結合組織中にその
構成成分として存在し、組織の柔軟性、構造維持、細胞
の代謝調節に重要な機能を果たしている。Hyaluronic acid is a type of polysaccharide that exists as a component in connective tissues such as bones, joints, vitreous body, zona, cartilage, skin, and the crest of birds. It plays an important regulatory function.
また、ヒアルロン酸のナトリウム塩（以下、ヒアルロン
酸ナトリウムという。）は、高分子物質であり、その溶
液は強い粘弾性をもち、保水作用を有するところから、
化粧品原料として広く使用されるほか、眼科治療薬、目
薬、関節症治療薬としての用途がある。In addition, sodium salt of hyaluronic acid (hereinafter referred to as sodium hyaluronate) is a polymeric substance, and its solution has strong viscoelasticity and has a water-retaining effect.
In addition to being widely used as a raw material for cosmetics, it also has uses as an ophthalmological treatment, eye drops, and arthropathy treatment.
（従来の技術）
ヒアルロン酸ナトリウムは工業的に、ニワトリのトサカ
、牛の目の硝子体もしくは屓帯等から抽出したヒアルロ
ン酸をそのナトリウム塩として採取する方法（抽出法）
やヒアルロン酸産生能を有する微生物を培地に培養し、
生成ヒアルロン酸をそのナトリウム塩として採取する方
法（醗酵法）によって得られている。(Prior art) Sodium hyaluronate is produced industrially by extracting hyaluronic acid from the crest of a chicken, the vitreous body or ligament of a cow's eye, etc. as its sodium salt (extraction method).
and microorganisms capable of producing hyaluronic acid are cultured in a medium,
It is obtained by a method (fermentation method) in which produced hyaluronic acid is collected as its sodium salt.
上述の抽出法や醗酵法によシ得られたヒアルロン酸ナト
リウムは、溶剤沈殿、イオン交換樹脂処理、分別沈殿、
活性炭処理などの方法によって精製される。従来、粉末
状ヒアルロン酸ナトリウムの製造法としては、該精製ヒ
アルロン酸ナトリウムを水に溶解させてヒアルロン酸ナ
トリウムの水溶液とし、該水溶液をスプレー乾燥する方
法、該水溶液を凍結乾燥する方法、精製ヒアルロン酸ナ
トリウムを塩化ナトリウムを含む水に溶解した水溶液に
水溶性有機溶剤を加えてヒアルロン酸ナトリウムを沈殿
させ、得られた沈殿物を母液と分離したのち、さらに水
溶性有機溶剤で洗浄することなく得られた該沈殿物をそ
のまま真空乾燥する方法、分離後の該沈殿物をさらに水
溶性有機溶剤で洗浄したのち該沈殿物を凍結乾燥する方
法（特開昭６２−２８９１９７号公報）などが知られて
いる。Sodium hyaluronate obtained by the above-mentioned extraction method or fermentation method is subjected to solvent precipitation, ion exchange resin treatment, fractional precipitation,
It is purified by methods such as activated carbon treatment. Conventionally, methods for producing powdered sodium hyaluronate include a method of dissolving the purified sodium hyaluronate in water to obtain an aqueous solution of sodium hyaluronate, and spray drying the aqueous solution, a method of freeze-drying the aqueous solution, and a method of freeze-drying the aqueous solution. A water-soluble organic solvent is added to an aqueous solution in which sodium is dissolved in water containing sodium chloride to precipitate sodium hyaluronate, and the resulting precipitate is separated from the mother liquor, which is then obtained without further washing with a water-soluble organic solvent. A method of vacuum drying the precipitate as it is, a method of washing the precipitate after separation with a water-soluble organic solvent, and then freeze-drying the precipitate (Japanese Patent Laid-Open No. 62-289197) are known. There is.
（発明が解決しようとする課題）
しかしながら、ヒアルロン酸ナトリウム水溶液をスプレ
ー乾燥する方法では乾燥時の熱によシ、得られるヒアル
ロン酸ナトリウムが低分子化し、かつ、乾燥装置が大が
かりなものになるといった欠点があシ、また該水溶液を
凍結乾燥する方法では、得られた粉末状ヒアルロン酸ナ
トリウムが綿状となって嵩密度が非常に小さくなシ、該
綿状のヒアルロン酸ナトリウム粉末を再度水に溶解させ
るときにままこになって溶解に長時間を要するといった
大きな欠点がある。(Problems to be Solved by the Invention) However, in the method of spray drying a sodium hyaluronate aqueous solution, the resulting sodium hyaluronate has a low molecular weight due to heat during drying, and the drying equipment becomes large-scale. However, in the method of freeze-drying the aqueous solution, the obtained powdered sodium hyaluronate becomes flocculent and has a very low bulk density. It has a major disadvantage that it becomes lumpy when dissolved and takes a long time to dissolve.
また、沈殿物を真空乾燥する方法では、該真空乾燥時に
粉末状の沈殿物が固化し、塊状化してしまい、再度水に
溶解させる際に水に溶解しにくいものＫなってしまうと
いった欠点がある。In addition, the method of vacuum drying the precipitate has the disadvantage that the powdered precipitate solidifies and becomes agglomerated during the vacuum drying, and when it is dissolved in water again, it becomes a substance that is difficult to dissolve in water. .
沈殿物を凍結乾燥する方法は、比較的水への溶解性のよ
い粉末状ヒアルロン酸ナトリウムが得られるが、−１０
℃以下の温度で高真空下に乾燥しなければならず、乾燥
に長時間を要し、その操作が繁雑であるとともに電力コ
ストが高くなるといった欠点がある。The method of freeze-drying the precipitate yields powdered sodium hyaluronate with relatively good solubility in water, but -10
Drying must be carried out under a high vacuum at a temperature below 0.degree. C., which requires a long time to dry, and has the drawbacks of complicated operations and high power costs.
従来よシ、安いコストで、水への溶解性の良好な粉末状
ヒアルロン酸ナトリウムを製造する方法の開発が要望さ
れていた。Conventionally, there has been a demand for the development of a method for producing powdered sodium hyaluronate with good solubility in water at low cost.
本発明者らは、簡単な操作で、コストが安く、水への溶
解性の良好な粉末状ヒアルロン酸ナトリウムを製造する
方法について鋭意研究した。その結果、塩化ナトリウム
含有のヒアルロン酸ナトリウム水溶液に、水溶性有機溶
剤を加えてヒアルロン酸ナトリウムを沈殿させ、該沈殿
物を母液と分離したのち、該沈殿物を再度、水溶性有機
溶剤で洗浄して、該沈殿物中の残存水分を除去し、得ら
れた沈殿物を減圧乾燥するととくよ）、コストが安く、
しかも水への溶解性に優れた粉末状ヒアルロン酸ナトリ
ウムが得られることを見い出し、本発明を完成した。The present inventors have conducted extensive research into a method for producing powdered sodium hyaluronate that is simple, inexpensive, and has good solubility in water. As a result, a water-soluble organic solvent was added to an aqueous sodium hyaluronate solution containing sodium chloride to precipitate sodium hyaluronate, and the precipitate was separated from the mother liquor, and then the precipitate was washed again with a water-soluble organic solvent. The remaining moisture in the precipitate is removed, and the obtained precipitate is dried under reduced pressure), the cost is low,
Moreover, they discovered that powdered sodium hyaluronate with excellent water solubility can be obtained, and completed the present invention.
以上の記述から明らかなように、本発明の目的は、安価
でしかも水への溶解性の良好な粉末状ヒアルロン酸ナト
リウムを製造する方法を提供することである。As is clear from the above description, an object of the present invention is to provide a method for producing powdered sodium hyaluronate that is inexpensive and has good solubility in water.
（課題を解決するための手段） 本発明は下記の構成を有する。(Means for solving problems) The present invention has the following configuration.
（１）塩化ナトリウム含有のヒアルロン酸ナトリウム水
溶液に水溶性有機溶剤を加えて、ヒアルロン酸ナトリウ
ムを沈殿させ、該沈殿物を母液と分離したのち、該沈殿
物を水溶性有機溶剤で洗浄して該沈殿物中の残存水分を
除去し、得られた沈殿物を減圧乾燥することを特徴とす
る粉末状ヒアルロン酸ナトリウムの製造法。(1) Add a water-soluble organic solvent to a sodium hyaluronate aqueous solution containing sodium chloride to precipitate sodium hyaluronate, separate the precipitate from the mother liquor, and then wash the precipitate with a water-soluble organic solvent to remove the sodium hyaluronate. A method for producing powdered sodium hyaluronate, which comprises removing residual water in the precipitate and drying the obtained precipitate under reduced pressure.
（２）沈殿物中の残存水分を水溶性有機溶剤で除去する
ための洗浄方法として、該沈殿物と該水溶性有機溶剤と
を撹拌混合したのち、該沈殿物を沈降させて上澄み液を
除去する方法もしくは該沈殿物上に該水溶性有機溶剤を
散布して、吸引濾過する衷法を用いる前記第１項に記載
の粉末状ヒアルロン酸ナトリウムの製造方法。(2) As a cleaning method for removing residual moisture in the precipitate with a water-soluble organic solvent, the precipitate and the water-soluble organic solvent are stirred and mixed, and then the precipitate is allowed to settle and the supernatant liquid is removed. 2. The method for producing powdery sodium hyaluronate according to item 1 above, which uses a method of spraying the water-soluble organic solvent on the precipitate and filtering it with suction.
（３）水溶性有機溶剤として、メタノール、エタノール
、アセトン、ｎ−プロパノール、インプロパツール、ア
セトニトリルもしくはこれらの２種以上の混合物を用い
る前記第１項に記載の粉末状ヒアルロン酸ナトリウムの
製造方法。(3) The method for producing powdered sodium hyaluronate according to item 1 above, wherein methanol, ethanol, acetone, n-propanol, impropatol, acetonitrile, or a mixture of two or more thereof is used as the water-soluble organic solvent.
（４）沈殿物を母液と分離する方法として、該沈殿物を
沈降させて上澄み液を除去する方法、吸引濾過する方法
、加圧濾過する方法もしくは遠心分離する方法を用いる
前記第１項に記載の粉末状ヒアルロン酸ナトリウムの製
造法。(4) As the method for separating the precipitate from the mother liquor, the method described in the above item 1 uses a method of settling the precipitate and removing a supernatant liquid, a method of suction filtration, a method of pressure filtration, or a method of centrifugation. A method for producing powdered sodium hyaluronate.
本発明に用いるヒアルロン酸ナトリウムは、ヒアルロン
酸産生能を有する微生物を栄養培地に培養し 該培養液
よシ得られたものすなわち醗酵法によシ得られたものや
生体よシ抽出したヒアルロン酸をナトリウム塩にしたも
のすなわち抽出方法によシ得られたものなどである。ヒ
アルロン酸産生能を有する微生物としてはヒアルロン酸
を菌体外に分泌する菌株であれば使用できるが、ストレ
プトコツカス属の菌株たとえばストレプトコッカス・ピ
オゲネス、ストレプトコッカス・エクイ、ストレプトコ
ッカス・エクイシミリス、ストレプトコッカス・ディス
ガラクチイエ、ストレプトコッカス・ズーエピデミカス
などを用いるのが好ましい。醗酵法によシヒアルロン酸
ナトリウムを得る方法としては、上述のヒアルロン酸産
生菌をたとえば培養液１１当シブドウ糖２０〜３０，９
．酵母エキス５１１ リン酸１カルシウム３１１ リン
酸２カリウム２１１チオ硫酸ナトリウム０．１．９ 、
硫酸マグネシウム７水塩０． Ｉ Ｆ 、亜硫酸ナトリ
ウム０．０２ｇ、塩化コバルト０．０１！ｉ、塩化マン
ガン０．０１１！、消泡剤５．９ ｔ−含ｂｐＨ６，Ｏ
−８，５、好ましくは７．０の培養液中で培養温度２５
８Ｃ〜４０℃、好ましくは３０°Ｃ−３５℃で１〜３日
間振とり培養もしくは通気培養し、ヒアルロン酸を該培
養液中に生成蓄積させ、該培養液を遠心分離もしくは濾
過して菌体を除去したのち、Ｆ液を限外濾過もしくは透
析することＫよって低分子量物質を除去し、低分子量物
質を除去したＦ液に水溶性有機溶剤を添加してヒアルロ
ン酸ナトリウムを沈殿させ、ついで該沈殿物を臭化セチ
ルトリメチルアンモニウム等による分画沈殿、イオン交
換クロマトグラフィー処理、活性炭処理、（脱色処理）
濃縮工程を経て濃縮された精製ヒアルロン酸ナトリウム
水溶液とする方法を例示することができる。Sodium hyaluronate used in the present invention is obtained by culturing microorganisms capable of producing hyaluronic acid in a nutrient medium, that is, by fermentation, or by extracting hyaluronic acid from living organisms. These include those made into sodium salts, that is, those obtained by extraction methods. As microorganisms capable of producing hyaluronic acid, any strain that secretes hyaluronic acid to the outside of the cell can be used, including strains of the genus Streptococcus, such as Streptococcus pyogenes, Streptococcus equi, Streptococcus equisimilis, and Streptococcus dysgalacti. It is preferable to use housewater, Streptococcus zooepidemicus, and the like. As a method for obtaining sodium hyaluronate by a fermentation method, the above-mentioned hyaluronic acid-producing bacteria are mixed with 11 parts of a culture solution and 20 to 30,9% of cyglucose.
．． Yeast extract 511 Monocalcium phosphate 311 Dipotassium phosphate 211 Sodium thiosulfate 0.1.9,
Magnesium sulfate heptahydrate 0. I F , sodium sulfite 0.02g, cobalt chloride 0.01! i, manganese chloride 0.011! , antifoaming agent 5.9 t-containing bpH 6,0
- Culture temperature 25 in a culture medium of 8.5, preferably 7.0
Shaking culture or aerated culture is performed at 8C to 40C, preferably 30C to 35C for 1 to 3 days to produce and accumulate hyaluronic acid in the culture solution, and the culture solution is centrifuged or filtered to remove bacterial cells. After removing the F solution, low molecular weight substances are removed by ultrafiltration or dialysis, and a water-soluble organic solvent is added to the F solution from which the low molecular weight substances have been removed to precipitate sodium hyaluronate. The precipitate is subjected to fractional precipitation using cetyltrimethylammonium bromide, etc., ion exchange chromatography treatment, activated carbon treatment, (decolorization treatment)
An example is a method of obtaining a concentrated purified sodium hyaluronate aqueous solution through a concentration step.
つぎに本発明の粉末状ヒアルロン酸ナトリウムの製造法
について述べると、上述の濃縮された精製ヒアルロン酸
ナトリウム水溶液（水溶液１１当り、ヒアルロン酸ナト
リウム約５１含有）に０．２モル／ｌになるように塩化
ナトリウムを溶解させる。該塩化ナトリウム含有ヒアル
ロン酸ナトリウム水溶液を激しく撹拌しつつ、アセトン
、メタノール、エタノール、ｎ−７’ロバノール、ｉ−
プロパノール、アセトニトリルなどの水溶性有機溶剤を
徐々に添加する。該水溶性有機溶剤の添加・にっれてヒ
アルロン酸ナトリウムの沈殿が析出してくる。添加する
該水溶性有機溶剤の量はヒアルロン酸ナトリウム水溶液
の量の２〜４倍がよい。このとき用いる該水溶性有機溶
剤は実質的に水分を含まない無水のものが好ましい。該
水溶性有機溶剤の添加が終了したのち、撹拌を停止し、
沈殿物を母液と分離する。Next, to describe the method for producing powdered sodium hyaluronate of the present invention, the above-mentioned concentrated and purified sodium hyaluronate aqueous solution (contains about 51 sodium hyaluronate per 11 aqueous solution) is added to the powdered sodium hyaluronate so as to have a concentration of 0.2 mol/l. Dissolve the sodium chloride. While vigorously stirring the sodium chloride-containing sodium hyaluronate aqueous solution, acetone, methanol, ethanol, n-7' lovanol, i-
Gradually add a water-soluble organic solvent such as propanol or acetonitrile. Upon addition of the water-soluble organic solvent, sodium hyaluronate precipitates out. The amount of the water-soluble organic solvent to be added is preferably 2 to 4 times the amount of the sodium hyaluronate aqueous solution. The water-soluble organic solvent used at this time is preferably an anhydrous one that does not substantially contain water. After the addition of the water-soluble organic solvent is completed, stirring is stopped,
Separate the precipitate from the mother liquor.
該分離法としては、沈殿物を沈降させてその上澄み液を
除去するいわゆるデカンテーション法、該沈殿物を含有
する母液を吸引濾過もしくは加圧濾過する方法、遠心分
離機にょシ遠心分離する方法などが用いられる。Examples of such separation methods include the so-called decantation method in which the precipitate is settled and the supernatant liquid is removed, a method in which the mother liquor containing the precipitate is subjected to suction filtration or pressure filtration, and a method in which centrifugation is performed using a centrifuge. is used.
次に１上述の分離法にょシ分離された沈殿物には水分が
残存しているので該残存水分を除去するために、該沈殿
物を水溶性有機溶剤で十分洗浄する。このとき用いる水
溶性有機溶剤としては、ヒアルロン酸ナトリウム水溶液
に添加して該ヒアルロン酸ナトリウムを沈殿させるとき
に用いた水溶性有機溶剤と同様、アセトン、メタノール
、エタノール、ｎ−プロパノール、ｉ−プロパノール、
アセトニトリルなどを例示することができ、該水溶性有
機溶剤は実質的に水分を含まない無水のものを用いるこ
とが好ましい。Next, since water remains in the precipitate separated by the above separation method, the precipitate is sufficiently washed with a water-soluble organic solvent to remove the residual water. The water-soluble organic solvents used at this time include acetone, methanol, ethanol, n-propanol, i-propanol, similar to the water-soluble organic solvents used when adding to the sodium hyaluronate aqueous solution to precipitate the sodium hyaluronate.
Examples include acetonitrile, and it is preferable to use an anhydrous one that does not substantially contain water as the water-soluble organic solvent.
該水溶性有機溶剤を用いて沈殿物中の残存水分を除去す
るだめの洗浄方法としては、■ヒアルロン酸ナトリウム
水溶液に添加して該ヒアルロン酸ナトリウムを沈殿させ
るときに用いたのと同量の水溶性有機溶剤を残存水分を
有する沈殿物に添加して１〜２０分間撹拌混合して、残
存水分を十分に該水溶性有機溶剤と置換したのち、該水
溶性有機溶剤と沈殿物とを分離して洗浄する方法、■残
存水分を有する沈殿物上にまんべんなく上述の洗浄方法
で用いたのと同量の洗浄用の水溶性有機溶剤を散布しな
がら、吸引濾過して該水溶性有機溶剤を分離して洗浄す
る方法などをあげることができる。As a cleaning method for removing residual moisture in the precipitate using the water-soluble organic solvent, 1. Add the same amount of aqueous solution as that used when precipitating the sodium hyaluronate by adding it to the sodium hyaluronate aqueous solution. The water-soluble organic solvent is added to the precipitate having residual moisture and stirred and mixed for 1 to 20 minutes to sufficiently replace the residual moisture with the water-soluble organic solvent, and then the water-soluble organic solvent and the precipitate are separated. (1) Spraying the same amount of the water-soluble organic solvent for cleaning as used in the above-mentioned cleaning method evenly over the precipitate containing residual water, and separating the water-soluble organic solvent by suction filtration. For example, how to clean it.
本発明の製造方法にあっては、沈殿物中の残存水分を除
去するための該洗浄が不可欠であり、該洗浄を実施しな
いときには、得られた沈殿物を減圧乾燥する段階で沈殿
物が固化、塊状化し、水への溶解性に優れた粉末状ヒア
ルロン酸ナトリウムが得られない。In the production method of the present invention, washing to remove residual moisture in the precipitate is essential, and if this washing is not performed, the precipitate will solidify during the step of drying the obtained precipitate under reduced pressure. , it becomes lumpy and powdered sodium hyaluronate with excellent water solubility cannot be obtained.
上述の■の方法で、用いた水溶性有機溶剤と沈殿物とを
分離する方法としては、ヒアルロン酸ナトリウム水溶液
に水溶性有機溶剤を添加して該ヒアルロン酸ナトリウム
を沈殿させ、沈殿物を母液と分離する際に用いたのと同
様の分離方法を用いればよい。To separate the water-soluble organic solvent used and the precipitate in the method (2) above, add a water-soluble organic solvent to an aqueous sodium hyaluronate solution to precipitate the sodium hyaluronate, and then mix the precipitate with the mother liquor. A separation method similar to that used for separation may be used.
水溶性有機溶剤で残存水分の除去のための洗浄が終了し
たのち、得られた沈殿物を減圧下に乾燥する。減圧乾燥
の条件としては、乾燥温度５０’Ｃ以下、圧力１トル以
下、乾燥時間５〜４８時間程度である。After washing with a water-soluble organic solvent to remove residual moisture is completed, the resulting precipitate is dried under reduced pressure. The conditions for drying under reduced pressure are a drying temperature of 50'C or less, a pressure of 1 Torr or less, and a drying time of about 5 to 48 hours.
かくして水への溶解性に優れた粉末状ヒアルロン酸ナト
リウムを製造することができる。In this way, powdered sodium hyaluronate with excellent water solubility can be produced.
（実施例）
以下、実施例および比較例を用いて本発明を具体的に説
明するが、本発明はこれによって限定されるものではな
い。なお、実施例および比較例で用いた粉末状ヒアルロ
酸ナトリウムの溶解性試験は次の方法によった。(Examples) Hereinafter, the present invention will be specifically explained using Examples and Comparative Examples, but the present invention is not limited thereto. The solubility test of the powdered sodium hyaluronate used in the Examples and Comparative Examples was conducted in the following manner.
１）溶解性試験
水ｘｏｏｍ／をビーカーに入れ、室温で１５゜ｒｐｍの
回転速度で撹拌しながら、ヒアルロン酸ナトリウムの試
料１００ｒｎ９を加え、該試料を加え終ってから溶解し
終るまでの時間を測定した。1) Put the solubility test water xoom/ into a beaker, add sample 100rn9 of sodium hyaluronate while stirring at a rotation speed of 15° rpm at room temperature, and measure the time from the time the sample is added until the time it completely dissolves. did.
実施例１
培養液ＩＩ当シ、ブドウ糖２０．Ｐ％酵母エキス５ｇ、
ペプトン１５ｙ、リン酸１カリウム３Ｎ。Example 1 Culture solution II, glucose 20. P% yeast extract 5g,
Peptone 15y, monopotassium phosphate 3N.
リン酸２カリウム２Ｉ！、チオ硫酸ナトリウム０．１１
、硫酸マグネシウム７水塩０．１９、亜硫酸ナトリウム
０．０２，９．塩化コバルト０．０１．？、塩化マンガ
ン０．０１Ｎ、大豆油５ＩからなるｐＨ７，０の培地１
．２１を内容積２１のミニジャーファーメンタ−に注入
し、１２０℃で２０分間加熱滅菌し、室温まで冷却した
のち、ストレプトコッカス・ズーエピデミカスの前培養
液０．１１を上述の培地に接種し、回転数３００ ｒｐ
ｍ、通気量Ｑ、 ７ ｖｖｍ 、温度３５°Ｃ，ｐＨを
７．０に自動制御して２４時間通気培養したのち、該培
養液にイオン交換水２．４１、ラジオライト４１００３
０Ｊｉ’を加えて、ヌツチェで濾過し、ν液を採取した
。Dipotassium phosphate 2I! , sodium thiosulfate 0.11
, magnesium sulfate heptahydrate 0.19, sodium sulfite 0.02,9. Cobalt chloride 0.01. ? , pH 7.0 medium 1 consisting of 0.01N manganese chloride and 5I soybean oil.
．． 21 into a mini-jar fermentor with an internal volume of 21, heat sterilized at 120°C for 20 minutes, and cooled to room temperature.The above medium was inoculated with 0.11 of a preculture of Streptococcus zooepidemicus, and the rotation speed was increased. 300rp
m, aeration amount Q, 7 vvm, temperature 35°C, pH automatically controlled to 7.0, aerated culture for 24 hours, and then ion-exchanged water 2.41 and Radiolite 41003 were added to the culture solution.
0Ji' was added and filtered through Nutsche to collect the ν solution.
該戸液３．５１を限界濾過機で１．５１に濃縮し、つい
で該濃縮液に３１のイオン交換水を加え、限界濾過機で
１．５１に濃縮する透析操作を３回くシ返した。該透析
終了後、濃縮液１．５ノに４重量％の臭化セチルトリメ
チルアンモニウム水溶液０．２ノを添加して、生成した
沈殿を分取し念。ついで該沈殿物を０．３モル／ｌの塩
化ナトリウム水溶液１．５１に溶解した。該溶液に粉末
活性炭６０９を加え、１時間撹拌したのち、ヌッチェで
濾過を行ない、精製ヒアルロン酸ナトリウム水溶液を得
た。The 3.51 solution was concentrated to 1.51 using an ultrafilter, then ion-exchanged water of 31 was added to the concentrated solution, and the dialysis operation of concentrating to 1.51 using an ultrafilter was repeated three times. . After the dialysis, 0.2 parts of a 4% by weight aqueous cetyltrimethylammonium bromide solution was added to 1.5 parts of the concentrate, and the precipitate formed was separated. The precipitate was then dissolved in 1.51 g of a 0.3 mol/l aqueous sodium chloride solution. Powdered activated carbon 609 was added to the solution, stirred for 1 hour, and then filtered through a Nutsche filter to obtain a purified aqueous sodium hyaluronate solution.
該精製ヒアルロン酸ナトリウム水溶液に、強い撹拌下で
無水のエタノール３１を、徐々に添加し、ヒアルロン酸
ナトリウムの沈殿を析出させ、該沈殿物と母液とを吸引
濾過法にて分離したのち、該沈殿物を２００１ｎｌの無
水のエタノール中に入れ、１０分間撹拌混合し、吸引濾
過法によって沈殿を分離して残存水分を除去した沈殿物
を得た。該沈殿物を温度４０°Ｃ１圧力１トル（’ｌ’
ｏｒｒ）以下で１２時間減圧乾燥を行ない、乾燥した粉
末状のヒアルロン酸ナトリウム２．６．９を得た。得ら
れた粉末状ヒアルロン酸す） ＩＪウムを用いて溶解性
試験を実施したところ、溶解時間は１０分であった。Anhydrous ethanol 31 was gradually added to the purified sodium hyaluronate aqueous solution under strong stirring to precipitate sodium hyaluronate, and the precipitate and mother liquor were separated by a suction filtration method. The mixture was placed in 2001 nl of anhydrous ethanol, stirred and mixed for 10 minutes, and the precipitate was separated by suction filtration to obtain a precipitate from which residual water had been removed. The precipitate was heated at a temperature of 40°C and a pressure of 1 Torr ('l'
orr) for 12 hours under reduced pressure to obtain a dried powdered sodium hyaluronate 2.6.9. A solubility test was conducted using the obtained powdered hyaluronic acid IJum, and the dissolution time was 10 minutes.
実施例２
実施例１に準拠して得た精製ヒアルロン酸ナトリウム水
溶液に、強い撹拌下で無水のエタノール３１を徐々に添
加し、ヒアルロン酸ナトリウムの沈殿を析出させ、該沈
殿物と母液とを吸引濾過法にて分離したのち、該沈殿物
を２００ｍ１の無水のアセトン中に入れ、実施例１に準
拠して残存水分の除去、吸引濾過、減圧乾燥して粉末状
のヒアルロン酸ナトリウム２．８１を得た。得られた粉
末状ヒアルロン酸ナトリウムを用いて溶解性試験を実施
したところ溶解時間は１０分であった。Example 2 Anhydrous ethanol 31 was gradually added to the purified sodium hyaluronate aqueous solution obtained according to Example 1 under strong stirring to precipitate sodium hyaluronate, and the precipitate and mother liquor were sucked. After separation by filtration, the precipitate was placed in 200 ml of anhydrous acetone, residual water was removed, suction filtrated, and dried under reduced pressure according to Example 1 to obtain powdered sodium hyaluronate 2.81. Obtained. A solubility test was conducted using the obtained powdered sodium hyaluronate, and the dissolution time was 10 minutes.
比較例１
実施例１に準拠して得た精製ヒアルロン酸ナトリウム水
溶液に、強い撹拌下で無水のエタノール３１を徐々に添
加し、ヒアルロン酸ナトリウムの沈殿を析出させ、該沈
殿物と母液とを吸引濾過法で分離したのち、該沈殿物を
水溶性有機溶剤で洗浄することなくそのまま減圧乾燥（
温度４０℃、圧力１トル（Ｔｏｒｒ） 、乾燥時間１２
時間）し、粉末状のヒアルロン酸ナトリウムを得た。得
られた粉末状ヒアルロン酸ナトリウムを用いて溶解性試
験を実施したところ、溶解時間は３０分であった。Comparative Example 1 Anhydrous ethanol 31 was gradually added to the purified sodium hyaluronate aqueous solution obtained according to Example 1 under strong stirring to precipitate sodium hyaluronate, and the precipitate and mother liquor were sucked. After separation by filtration, the precipitate was dried under reduced pressure (without washing with a water-soluble organic solvent).
Temperature: 40°C, pressure: 1 Torr, drying time: 12
time) to obtain powdered sodium hyaluronate. When a solubility test was conducted using the obtained powdered sodium hyaluronate, the dissolution time was 30 minutes.
比較例２
実施例１に準拠して得た精製ヒアルロン酸ナトリウム水
溶液に実施例工に準拠して無水エタノール３１を徐々に
添加してヒアルロン酸ナトリウムを沈殿させ、吸引濾過
法によって母液と分離して得た沈殿物を５００−の蒸留
水に溶解した。得られた該水溶液を圧力１トル（Ｔｏｒ
ｒ）以下で、１２時間凍結乾燥し、綿状のヒアルロン酸
ナトリウム２．３ｇを得た。得られたヒアルロン酸ナト
リウムの溶解性試験を実施したところ、溶解時間は４時
間であった。Comparative Example 2 Anhydrous ethanol 31 was gradually added to the purified sodium hyaluronate aqueous solution obtained according to Example 1 to precipitate sodium hyaluronate, and the sodium hyaluronate was separated from the mother liquor by a suction filtration method. The obtained precipitate was dissolved in 500-g distilled water. The resulting aqueous solution was heated to a pressure of 1 Torr.
r) The following was freeze-dried for 12 hours to obtain 2.3 g of flocculent sodium hyaluronate. When the obtained sodium hyaluronate was subjected to a solubility test, the dissolution time was 4 hours.
比較例３
実施例１に準拠して得たヒアルロン酸ナトリウム水溶液
を用いて、実施例１に準拠して、水溶性有機溶剤で洗浄
した沈殿物を得た。該沈殿物を一４０℃で凍結したのち
、棚温度を一２０℃に設定した乾燥機で１２時間凍結乾
燥し、ヒアルロン酸す） ＩＪウムの乾燥物２．６ｇを
得た。得られたヒアルロン酸ナトリウム乾燥物を乳鉢で
粉砕し、粉末状のヒアルロン酸ナトリウムとした。得ら
れた粉末状ヒアルロン酸ナトリウムを用いて溶解性試験
を実施したところ溶解時間は３０分であった。Comparative Example 3 Using the sodium hyaluronate aqueous solution obtained in accordance with Example 1, a precipitate was obtained which was washed with a water-soluble organic solvent in accordance with Example 1. The precipitate was frozen at -40°C and then freeze-dried for 12 hours in a dryer with a shelf temperature of -20°C to obtain 2.6 g of dried hyaluronic acid IJum. The obtained dried sodium hyaluronate was ground in a mortar to obtain powdered sodium hyaluronate. When a solubility test was conducted using the obtained powdered sodium hyaluronate, the dissolution time was 30 minutes.
実施例３
精製ヒアルロン酸ナトリウム水溶液として、抽出法によ
υ得られた市販のヒアルロン酸ナトリウム（キューピー
■製、ＨＡ−Ｑ）２．Ｆを０．３モル／ｌの塩化ナトリ
ウム水溶液１１に溶解したヒアルロン酸す） ＩＪウム
水溶液を用いる以外は実施例１に準拠して、ヒアルロン
酸ナトリウムの沈殿の析出、無水エタノールによる該沈
殿物の洗浄、減圧乾燥を行ない、１．７．９の粉末状ヒ
アルロン酸ナトリウムを得た。得られた粉末状ヒアルロ
ン酸ナトリウムの溶解試験を実施したところ溶解時間は
１０分であった。Example 3 Commercially available sodium hyaluronate (manufactured by Kewpie ■, HA-Q) obtained by an extraction method as a purified sodium hyaluronate aqueous solution.2. Hyaluronic acid in which F was dissolved in 0.3 mol/l sodium chloride aqueous solution After washing and drying under reduced pressure, powdered sodium hyaluronate of 1.7.9 was obtained. When the obtained powdered sodium hyaluronate was subjected to a dissolution test, the dissolution time was 10 minutes.
比較例４
実施例３に準拠して得たヒアルロン酸ナトリウム水溶液
に、強い撹拌下に無水のエタノール３１を徐々に添加し
てヒアルロン酸ナトリウムの沈殿を析出させ、該沈殿物
と母液とを吸引濾過法によシ分離したのち、該沈殿物を
水溶性有機溶剤で洗浄することなく、減圧乾燥（温度４
０℃、圧力１トル（Ｔｏｒｒ） 、乾燥時間１２時間）
し、粉末状のヒアルロン酸ナトリウムを得た。得られた
粉末状ヒアルロン酸ナトリウムを用いて溶解性試験を実
施したところ、溶解時間は３０分であった。Comparative Example 4 Anhydrous ethanol 31 was gradually added to the sodium hyaluronate aqueous solution obtained according to Example 3 under strong stirring to precipitate sodium hyaluronate, and the precipitate and mother liquor were filtered with suction. After separating the precipitate using a water-soluble organic solvent, the precipitate was dried under reduced pressure (temperature 4.
0°C, pressure 1 Torr, drying time 12 hours)
Then, powdered sodium hyaluronate was obtained. When a solubility test was conducted using the obtained powdered sodium hyaluronate, the dissolution time was 30 minutes.
比較例５
抽出法により得られた市販のヒアルロン酸ナトリウム（
キューピー■製、ＨＡ−Ｑ）２ｆ９を蒸留水４００１１
Ｉ９に溶解したヒアルロン酸ナトリウム水溶液を圧力１
トル（Ｔｏｒｒ）以下で１２時間凍結乾燥し、綿状のヒ
アルロン酸ナトリウム１．９Ｉ！を得た。得られた該綿
状のヒアルロン酸ナトリウムを用いて、溶解性試験を実
施したところ溶解時間は３時間であった。Comparative Example 5 Commercially available sodium hyaluronate obtained by extraction method (
Made by Kewpie ■, HA-Q) 2f9 with distilled water 40011
A sodium hyaluronate aqueous solution dissolved in I9 was heated to a pressure of 1
Freeze-dry at below Torr for 12 hours to form a flocculent form of sodium hyaluronate 1.9I! I got it. A solubility test was conducted using the obtained flocculent sodium hyaluronate, and the dissolution time was 3 hours.
比較例６
実施例３に準拠して得たヒアルロン酸ナトリウム水溶液
を用いて、実施例３に準拠して、水溶性有機溶剤で洗浄
し九沈殿物を得た。該沈殿物を一４０℃にて凍結したの
ち、棚温度−２０℃で１２時間凍結乾燥し、ヒアルロン
酸ナトリウムの乾燥物１．６ｇを得た。該乾燥物を乳鉢
で粉砕し、粉末状のヒアルロン酸ナトリウムとした。該
粉末状のヒアルロン酸ナトリウムを用いて溶解性試験を
実施したところ、溶解時間は３０分であった。Comparative Example 6 Using the sodium hyaluronate aqueous solution obtained in accordance with Example 3, washing was performed with a water-soluble organic solvent in accordance with Example 3 to obtain nine precipitates. The precipitate was frozen at -40°C and then freeze-dried at a shelf temperature of -20°C for 12 hours to obtain 1.6 g of dried sodium hyaluronate. The dried product was ground in a mortar to obtain powdered sodium hyaluronate. When a solubility test was conducted using the powdered sodium hyaluronate, the dissolution time was 30 minutes.
（発明の効果）
本発明の製造方法によれば水への溶解性に優れた粉末状
ヒアルロン酸ナトリウムを容易にかつ安価に製造するこ
とができる。(Effects of the Invention) According to the production method of the present invention, powdered sodium hyaluronate having excellent solubility in water can be produced easily and at low cost.
以上that's all
Claims (4)
溶液に水溶性有機溶剤を加えてヒアルロン酸ナトリウム
を沈殿させ、該沈殿物を母液と分離したのち、該沈殿物
を水溶性有機溶剤で洗浄して該沈殿物中の残存水分を除
去し、得られた沈殿物を減圧乾燥することを特徴とする
粉末状ヒアルロン酸ナトリウムの製造法。(1) Add a water-soluble organic solvent to a sodium hyaluronate aqueous solution containing sodium chloride to precipitate sodium hyaluronate, separate the precipitate from the mother liquor, and then wash the precipitate with a water-soluble organic solvent to precipitate the precipitate. A method for producing powdered sodium hyaluronate, which comprises removing residual moisture in the product and drying the obtained precipitate under reduced pressure.
ための洗浄方法として、該沈殿物と該水溶性有機溶剤と
を撹拌混合したのち、該沈殿物を沈降させて上澄み液を
除去する方法もしくは該沈殿物上に該水溶性有機溶剤を
散布して、吸引濾過する方法を用いる請求項１記載の粉
末状ヒアルロン酸ナトリウムの製造方法。(2) As a cleaning method for removing residual moisture in the precipitate with a water-soluble organic solvent, the precipitate and the water-soluble organic solvent are stirred and mixed, and then the precipitate is allowed to settle and the supernatant liquid is removed. 2. The method for producing powdered sodium hyaluronate according to claim 1, which uses a method of spraying the water-soluble organic solvent on the precipitate and filtering it with suction.
、アセトン、ｎ−プロパノール、イソプロパノール、ア
セトニトリルもしくはこれらの２種以上の混合物を用い
る請求項１記載の粉末状ヒアルロン酸ナトリウムの製造
方法。(3) The method for producing powdered sodium hyaluronate according to claim 1, wherein methanol, ethanol, acetone, n-propanol, isopropanol, acetonitrile, or a mixture of two or more thereof is used as the water-soluble organic solvent.
沈降させて上澄み液を除去する方法、吸引濾過する方法
、加圧濾過する方法もしくは遠心分離する方法を用いる
請求項１記載の粉末状ヒアルロン酸ナトリウムの製造法
。(4) The powder according to claim 1, wherein the method for separating the precipitate from the mother liquor is a method of settling the precipitate and removing a supernatant liquid, a method of suction filtration, a method of pressure filtration, or a method of centrifugation. A method for producing sodium hyaluronate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14536888A JPH01313502A (en) | 1988-06-13 | 1988-06-13 | Production of powdery sodium hyaluronate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14536888A JPH01313502A (en) | 1988-06-13 | 1988-06-13 | Production of powdery sodium hyaluronate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01313502A true JPH01313502A (en) | 1989-12-19 |
Family
ID=15383601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14536888A Pending JPH01313502A (en) | 1988-06-13 | 1988-06-13 | Production of powdery sodium hyaluronate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01313502A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008512996A (en) * | 2004-09-23 | 2008-05-01 | バイエル・クロップサイエンス・アーゲー | Methods and means for producing hyaluronan |
| CN109851822A (en) * | 2018-12-07 | 2019-06-07 | 山东众山生物科技有限公司 | A kind of preparation method of instant Sodium Hyaluronate |
-
1988
- 1988-06-13 JP JP14536888A patent/JPH01313502A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008512996A (en) * | 2004-09-23 | 2008-05-01 | バイエル・クロップサイエンス・アーゲー | Methods and means for producing hyaluronan |
| CN109851822A (en) * | 2018-12-07 | 2019-06-07 | 山东众山生物科技有限公司 | A kind of preparation method of instant Sodium Hyaluronate |
| CN109851822B (en) * | 2018-12-07 | 2022-02-11 | 山东众山生物科技有限公司 | Preparation method of instant sodium hyaluronate |
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